Noncovalent functionalization of mesoporous
silica nanoparticles with amphiphilic peptides†
Melis Sardan,‡abAdem Yildirim,‡*abDidem Mumcuoglu,abAyse B. Tekinay*ab and Mustafa O. Guler*ab
The surface of mesoporous silica nanoparticles (MSNs) has been modified for enhancing their cellular uptake, cell targeting, bioimaging, and controlled drug release. For this purpose, covalent anchorage on the silica surface was predominantly exploited with a wide range of bioactive molecules. Here, we describe a facile self-assembly method to prepare a hybrid peptide silica system composed of octyl-modified mesoporous silica nanoparticles (MSNs) and peptide amphiphiles (PAs). The hydrophobic organosilane surface of mesoporous silica was coated with amphiphilic peptide molecules. The peptide functionalized particles exhibited good cyto-compatibility with vascular smooth muscle and vascular endothelial cells. The peptide coating also improved the cellular uptake of particles up to 6.3 fold, which is promising for the development of highly efficient MSN based theranostic agents.
Introduction
Mesoporous silica nanoparticles (MSNs) have high specic surface area, large pore volume, controlled particle size and morphology, and low inherent toxicity. They are promising platforms for theranostic applications.1–3 Previously, in vitro studies of MSNs demonstrated that these materials can be used as drug delivery and cell marking agents.4–6 However, it was observed that the bare silica surface (containing reactive silanol groups) can cause aggregation of particles and opsonization in a biological environment, and nonspecically interact with the membrane of several cell types, which results in poor biocom-patibility and pharmacokinetics for in vivo applications.7–11 Modication of the reactive MSN surface with polymers12–15 (e.g. PEG and zwitterionic copolymers), small molecules10,16(e.g. phosphonate) or biomolecules17–19 (e.g. antibodies, peptides, lipid bilayers, DNA and aptamers) has been proved to be an effective way to improve the biocompatibility of particles as well as the efficiency of MSN based therapies. In particular, short peptide chains have attracted a great deal of attention in recent years because of their tunable functionality, biodegradability, and relative ease of synthesis.20–22It has been shown that short peptide sequences (e.g. RGD and IL-13) can be used for targeting nanoparticles to specic cancer cell lines.22–26 In addition to
targeting properties, some peptide sequences (e.g. TAT peptide) demonstrated cell penetrating and endosomal escape proper-ties.27–29 Also, the biodegradability of peptides makes them suitable for being utilized as stimuli responsive gatekeepers in controlled drug release30,31 and linkers in FRET based diagnostics.32–34
Conventionally, peptides are covalently attached to the silica surface by using additional cross-linking reagents and troublesome synthetic methods, which results in poor surface graing density and costly synthesis of functional-ized materials.24,30,35In this work, we demonstrate a simple and cost-effective self-assembly approach to prepare peptide functionalized MSNs, as a promising alternative for covalent attachment methods.13Our method is based on the sponta-neous attachment of peptide amphiphiles (PAs) to the octyl modied MSN surfaces in aqueous media through hydro-phobic interactions between octyl groups of MSNs and alkyl chains of PAs (Scheme 1a). We selected two model PA mole-cules, with different charges, to functionalize MSNs. The glutamic acid and lysine residues on the PAs provide negative and positive charges to the hybrid system, respectively (Scheme 1b). The unmodied MSNs were synthesized in order to compare with peptide functionalized MSNs. The effect of peptide functionalization on cell viability and uptake was investigated by using human umbilical vein endothelial cells (HUVECs) and a vascular smooth muscle cell line (A10). All of the MSN systems demonstrated good cyto-compatibility with both cell lines up to a concentration of 200 mg mL1. Interestingly, we observed a remarkable increase (1.8 to 6.3 fold) in the cellular uptake of peptide functionalized MSNs compared to bare MSNs depending on the surface charge of PAs as well as the cell type.
aNational Nanotechnology Research Center (UNAM), Bilkent University, 06800 Ankara,
Turkey. E-mail: moguler@unam.bilkent.edu.tr; atekinay@unam.bilkent.edu.tr; Fax: +90 312 266 4365; Tel: +90 312 290 3552
bInstitute of Materials Science and Nanotechnology, Bilkent University, 06800 Ankara,
Turkey. E-mail: ademy@bilkent.edu.tr
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4tb00037d
‡ These authors contributed equally. Cite this:J. Mater. Chem. B, 2014, 2, 2168 Received 7th January 2014 Accepted 6th February 2014 DOI: 10.1039/c4tb00037d www.rsc.org/MaterialsB
Materials Chemistry B
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Experimental section
Materials9-Fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc) protectedL-amino acids, [4-[a-(20,40-dimethoxyphenyl) Fmocami-nomethyl]phenoxy]acetamidonorleucyl-MBHA resin (Rink amide MBHA resin), and 2-(1H-Benzotriazol-1-yl)-1,1,3,3 tetramethy-luronium hexauorophosphate (HBTU) were purchased from NovaBiochem and ABCR. Tetraethyl orthosilicate (TEOS), octyl-triethoxysilane (OTS), aminopropyl octyl-triethoxysilane (APTES), sodium hydroxide, and lauric acid were purchased from Merck. Fluorescein isothiocyanate (FITC), cetyltriammoniumbromide (CTAB) and ethanol were purchased from Sigma-Aldrich. Meth-anol was purchased from Carlo-Erba. Tetrahydrofuran (THF) was purchased from Labkim. All reagents and solvents were used as provided. Cell culture chemicals were purchased from Gibco, Life Technologies.
Synthesis of MSNs and OMSNs
In order to synthesize FITC labelled OMSNs, 2 mg of FITC was conjugated to 10mL of APTES in 1 mL of ethanol by gently stirring for 24 h. Then, 200 mg of CTAB and 6 mg of F127 were dissolved in 96 mL of deionized water and 0.7 mL of 2 M NaOH was added. The solution was heated to 80C under vigorous stirring (600 rpm). Aer the temperature of the reaction mixture was stabilized at 80C, 1 mL of TEOS and FITC solution was rapidly added. Aer 75 min, 0.2 mL of OTS was dissolved in 10 mL THF and slowly added to the reaction mixture in order to form an octyl-containing shell. The mixture was further stirred for 90 min. Finally, the particles were collected by centrifuga-tion at 9000 rpm and washed with methanol twice. Surfactant extraction was performed by stirring the particles in 50 mL of 20 g L1 ethanolic ammonium nitrate at 60 C for 1 h. This treatment was repeated twice to ensure complete surfactant
removal. Particles were washed with ethanol twice and dried at 50 C overnight. MSNs were synthesized by using the same parameters with the exception of OTS addition.
Synthesis and characterization of peptide amphiphile molecules
The positively charged peptide amphiphile (K-PA) was con-structed on MHBA Rink Amide (0.59 mmol g1loading) resin, and the negatively charged peptide amphiphile (E-PA) was con-structed on Fmoc-Glu-Wang (0.64 mmol g1loading) resin. All amino acid couplings were performed with 2 equivalents of Fmoc protected amino acid, 1.95 equivalents of HBTU and 3 equiva-lents of N,N-diisopropylethylamine (DIEA) in dimethylforma-mide (DMF) for 2 h. Fmoc removals were performed with 20% piperidine–DMF solution for 20 min. The cleavage of the peptides from the resin was carried out with a mixture of tri-uoroacetic acid (TFA)–triisopropylsilane (TIS)–water in the ratio of 95 : 2.5 : 2.5 for 2 h. Excess TFA was removed by rotary evap-oration. The remaining viscous peptide solution was treated with ice-cold diethyl ether and the resulting white pellet was freeze-dried. The peptide amphiphiles were identied and analyzed by reverse phase HPLC on an Agilent 6530 accurate-Mass Q-TOF LC/ MS equipped with an Agilent 1200 HPLC. A phenomenex Luna 3m C8 100A (50 3.00 mm) column as the stationary phase and water–acetonitrile gradient with 0.1% volume of formic acid as the mobile phase were used to identify the positively charged peptide amphiphile. For the negatively charged peptide amphi-phile, an Agilent Zorbax Extend-C18 (2.1 50 mm) column as the stationary phase and water–acetonitrile gradient with 0.1% volume of ammonium hydroxide as the mobile phase were used. The positively and negatively charged peptide amphiphiles were puried by using a 1200 Agilent HPLC on a Zorbax 300SB C8 (21.2 150 mm) PrepHT and a Zorbax-Extend C18 (21.2 150 mm) PrepHT column, respectively.
Coating the OMSN with peptide amphiphiles
While 14 mg of each peptide amphiphile was sonicated in 12 mL deionized water, 2 mg of OMSNs was slowly added to the system. Peptide amphiphiles and OMSNs were mixed in a weight ratio of 1 : 7. They were sonicated and vortexed for 3 h at room temperature. The solution was centrifuged at 5000 rpm. Aer all portions were concentrated by centrifugation, they were rinsed with water and centrifuged twice.
Characterization of the particles
The hydrodynamic size and zeta potential of the particles were measured by using a Zetasizer. A Malvern Nanosizer/Zetasizer nano-ZS ZEN 3600 (Malvern Instruments, USA) instrument was used for the analysis. Measurements were performed in glass cuvettes and repeated at least three times. TEM images were obtained using a FEI Tecnai G2 F30 TEM at 300 kV. Samples for imaging were prepared by diluting PA coated MSNs to 0.01% (w/v) on a 200-mesh copper TEM grid for 5 min without staining and air dried. Fluorescence spectra of the particles were recorded using a Varian Eclipse spectrophotometer with an excitation wavelength of 488 nm. FTIR spectra of particles and PAs were collected by Scheme 1 (a) Schematic presentation of the preparation of peptide
functionalized MSNs. (b) Chemical structures of the peptide amphi-phile molecules used in this study.
using an FTIR spectrometer (Vertex 70, Bruker). Thermogravi-metric analyses (TGA) were performed with a Q500 (TA Instru-ments). The temperature was increased from room temperature to 800C with a rate of 20C min1under nitrogen gas.
Cell culture experiments
Viability and uptake experiments were performed by using human umbilical vein endothelial cells (HUVECs) and A10 rat aortic smooth muscle cells (ATCC® Cat# CRL-1476™). HUVECs were donated by Yeditepe University, Istanbul, Turkey. HUVECs were puried as described36and characterized by staining with CD34, CD31, and CD90 surface markers. These cells were found to be positive for CD31 and CD34 but negative for CD90. A10 and HUVEC cells were cultured in 75 cm2 polystyrene cell culture asks with standard medium, containing Dulbecco's modied eagle medium (DMEM) with 10% fetal bovine serum (FBS), and 1% penicillin–streptomycin and passaged at cell conuency between 80 and 90% using trypsin–EDTA. In all experiments, particles were administered in serum free medium (1% peni-cillin–streptomycin containing DMEM) to avoid any inuence of serum proteins on the uptake mechanism.
Cell viability tests
The cell viability assay was performed using Alamar Blue Assay (Invitrogen). 5000 cells per well (HUVECs or A10) were seeded on a 96-well plate in 100mL of standard medium. Aer 24 h, the medium was removed and 100mL of serum free medium was added. 25mL of freshly prepared nanoparticle solutions in water at different concentrations were administered to have nal concentrations of 200, 100, 50, and 10mg mL1. For cell viability tests, cells were exposed to particles for 4 h, then the medium was changed to standard medium and the cells were further incubated for 20 h. Then, Alamar Blue reagent diluted to 10% in DMEM was added. Aer 3 h, uorescence at 570/612 nm (Ex/ Em) was measured using a microplate reader (SpectraMax, M5). Cellular uptake analyses
13 mm glass coverslips were placed in 24-well plates and 4 104 cells (HUVECs or A10) in standard medium were seeded in each well. Aer 24 h, the medium was discarded, and 400 mL of serum free medium was added to each well. 100mL of bare or functionalized MSNs were administered to have a nal concentration of 200mg mL1MSNs. Aer 4 h, the medium was exchanged to standard medium. Aer 20 h, the cells were washed with PBS several times and xed with 4% para-formaldehyde for 15 min. Then, the cells were permeabilized with 0.1% TritonX-100 (Sigma-Aldrich) for 12 min and actin proteins were stained with Phalloidin-TRITC (Sigma-Aldrich) for 20 min. Coverslips were mounted on slides with Antifade (Invitrogen). Samples were visualized with a laser scanning confocal microscope (Zeiss, LSM 510).
Flow cytometry analysis
1 105 HUVECs or A10 cells per well were seeded in 6 well plates in standard medium. The medium was discarded aer
24 h and 1600mL of serum free medium was added to each well. 400mL of bare or functionalized MSNs were administered to have anal concentration of 200 mg mL1MSNs. Aer 4 h, the medium was changed to standard medium and the cells were incubated for 20 h, then washed with PBS and trypsinized. The cells were collected by centrifugation, washed twice with PBS, resuspended in 1 mL PBS and kept on ice before analysis. An FITC channel was used to analyze the MSN uptake using aow cytometer (BD, FACS Aria III). Non-treated cells were used as the control. Student's t-test was applied to all datasets and a p value of less than 0.05 was accepted to be statistically signicant.
Results and discussion
Synthesis and characterization of peptide functionalized MSNs
Octyl modied water insoluble MSNs (OMSNs) were synthesized according to our previous reports13,37 by using a one-pot respective condensation method.38 Tetraethyl orthosilicate (TEOS) molecules were condensed under basic conditions and initial MSNs were formed. Then, octyl triethoxysilane (OTS) molecules were added to the reaction mixture to coat the MSNs with a hydrophobic octyl layer. Theuorescein isothiocyanate (FITC) molecules were conjugated to the MSNs in therst step of the synthesis to track the uptake of particles by using confocal imaging and ow cytometry methods.16 Successful conjugation of FITC to the silica network was demonstrated by usinguorescence spectroscopy, where emission bands of FITC molecules were clearly observed (ESI,† Fig. S1). Bare MSNs were prepared for control experiments under the same experimental conditions without the OTS addition. Peptide amphiphiles were synthesized by solid phase peptide synthesis method based on orthogonal protection and veried by liquid chromatography and mass spectrometry (ESI,† Fig. S2 and S3).
OMSNs were coated with peptide amphiphile molecules by simple sonication in E-PA or K-PA solutions (Fig. 1a). As-prepared OMSNs are insoluble in water (Fig. 1a, le) because their surface is covered with hydrophobic octyl groups. Aer the addition of PA solution and ultrasonication, PA molecules self-assemble on the OMSNs due to hydrophobic interactions between alkyl chains of both OMSNs and PAs (Scheme 1a). Functionalization with peptide amphiphiles renders the MSNs
Fig. 1 (a) Photograph showing the water dispersion of OMSNs before and after coating with peptide amphiphiles and (b) photographs of MSN dispersion in water.
water dispersible by providing either positively or negatively charged water soluble moieties on their surfaces. Photographs of MSNs dispersed in water are shown in Fig. 1b. Both disper-sions have light green color due to the covalently conjugated FITC molecules. Fig. 2 shows the TEM images of the particles. Bare MSNs exhibited an MCM-41 type highly ordered hexagonal porous structure (Fig. 2a). On the other hand, for OMSNs, a randomly porous thin shell was observed over an MCM-41 type porous core (Fig. 2b) which is in accordance with our previous work.13Aer coating the OMSNs with E-PAs, a thin organic layer was observed around the particles (Fig. 2c). In addition, a TEM image of K-PA peptide coated OMSNs is provided in the ESI† (Fig. S4).
The formation of a peptide coating over OMSNs was further proved by using FTIR and TGA methods. Fig. 3a shows the FTIR spectra of the particles. The–CH peaks between 2800 cm1and 3000 cm1were observed in the spectrum of OMSNs indicating the successful octyl modication. These absorption bands became stronger for PA modied particles (E-OMSNs and K-OMSNs) due to the–CH bonds of the PAs. In addition, new absorption bands between 1400 cm1and 1800 cm1appeared, which are in good agreement with FTIR spectra of the PAs. The amide I (1600–1690 cm1, C]O stretching) and amide II (1480–
1575 cm1, CN stretching, NH bending) bands of PAs were observed for PA functionalized MSNs. Another C]O stretching vibration was also observed for glutamic acid containing particles at around 1725 cm1 which corresponds to the side chain of the glutamic acid.
Fig. 3b shows the TGA analysis of the particles. For MSNs, a small weight loss of 7.8% was observed between 100C and 800C due to the dehydroxylation of the silica surface.39The weight loss increased to 11.9% for OMSNs and most of the weight loss occurred at around 500C indicating the decom-position of octyl moieties.13For E-OMSNs and K-OMSNs large weight loss values of 19.6% and 32.5% were recorded, respec-tively. Also, two sharp decreases were observed in the spectra of peptide coated particles around 400 C and 500 C, which correspond to the decomposition of PAs and octyl groups, respectively. For bare peptide amphiphiles, almost all weight was lost at 800 C. Based on the TGA results, we calculated graing densities of the PA molecules on the OMSN surfaces (see ESI† for details).40,41The graing densities of E-OMSNs and K-OMSNs were 0.94 and 2.95 PA nm2, respectively, indicating that the surface of OMSNs was densely covered by the peptide molecules. A higher graing density of K-OMSNs was observed due to the electrostatic interactions between positively charged
K-PAs and unreacted surface silanol groups of OMSNs. Particle sizes of the MSNs and OMSNs were calculated to be 100 nm and 104 nm, respectively from the TEM images (Table 1). The 4 nm increase in the particle size aer octyl addition shows the 2 nm shell formation around the particles. Hydrodynamic sizes of the MSN, E-MSN and K-MSN were measured by using the dynamic light scattering technique (Table 1 and ESI,† Fig. S5). The OMSNs are water insoluble and it is not possible to measure their hydrodynamic size. Hydrodynamic sizes of the peptide coated particles are slightly larger (around 40 nm) than their primary particle sizes (size of OMSNs) due to the slight
Fig. 2 Characterization of the mesoporous silica nanoparticles. TEM images of (a) MSNs, (b) OMSNs and (c) E-OMSNs.
Fig. 3 FTIR spectra (a) and TGA spectra (b) of particles and peptide amphiphiles, respectively.
Table 1 Physical properties of bare, octyl modified and peptide functionalized MSNs
Sample TEM size (nm) DLS size (nm) Zeta potential (mV)
MSNs 99.8 20.7 166.5 8.0 36.6 1.1
OMSNs 104.3 21.5 N/A N/A
E-OMSNs N/A 143.8 19.8 38.0 0.8
K-OMSNs N/A 145.2 1.6 25.1 0.7
aggregation in aqueous media for these particles. On the other hand, aggregation becomes more pronounced for bare MSNs with a 67 nm difference between hydrodynamic and primary particle sizes. This difference could be explained by the fact that two different techniques, TEM and DLS, were used for the measurements. While dried samples were used for TEM imaging, the hydrodynamic size of particles was measured using DLS where interactions with solvent molecules are also taken into account. The surfaces of the particles were charac-terized by measuring their zeta potentials (Table 1). The bare MSN surface is negatively charged (37 mV) due to the surface silanol groups.10Modifying the MSN surface with a negatively charged E-PA did not signicantly affect the zeta potential of the surface (38 mV) since both silanol and E-PA groups are negatively charged. Coating the MSN surface with a positively charged K-PA, on the other hand, resulted in a remarkable increase in the zeta potential (25 mV).
Cyto-compatibility of MSNs
A good compatibility of therapeutic nanoparticles with biolog-ical organisms is necessary to prevent possible side effects of
these therapies. Accordingly, the in vitro cytocompatibilities of the peptide coated and bare MSNs were evaluated by using HUVEC and A10 cell lines. The viability of the cells treated with different particle concentrations (10 to 200 mg mL1) was
studied by using the Alamar blue assay. First, cells were treated with particles for 4 h and then incubated for additional 20 h in particle free media. None of the particles showed decrease in the viability of both cell lines even at a very high particle concentration of 200mg mL1(Fig. 4a and b).
Cellular uptake studies
Improving the cellular uptake of the therapeutic nanoparticles is essential for enhancing their efficacy. Therefore, we studied the uptake of the particles by A10 cells and HUVEC by using confocal microscopy andow cytometry techniques. Both A10 cells and HUVEC were treated with 200mg mL1nanoparticles for 4 h and the cells were analyzed aer 20 h of further incu-bation. As shown in Fig. 5, confocal microscopy revealed that the peptide coated particles were internalized remarkably more than the bare MSNs by both cell lines.
In order to compare the uptake amount of the particles, the uorescence of the internalized particles was quantied by using aow cytometer (Fig. 6). More uptake was observed for K-OMSNs in both cell lines. The amount of internalized uo-rescent E-OMSNs was less than that of K-OMSNs, however, they were internalized more than bare particles. The uptake of K-OMSNs by A10 cells and HUVEC was 2.3 and 6.3 fold larger than the uptake of MSNs, respectively. Also, E-OMSNs demon-strated 1.8 and 3.1 fold increased uptake by A10 cells and
Fig. 5 Uptake results of bare and peptide functionalized MSNs. Confocal results showing that peptide functionalized particles were internalized more both A10 and HUVEC cell lines. Upper images at left show the fluorescence of particles, lower images at left show the fluorescence of actin filaments stained by Phalloidin-TRITC and panels on right show the merged images.
Fig. 4 Cytotoxicity results of bare and peptide functionalized MSNs. (a) A10 cells incubated for 4 h with particles and 20 h in particle free media and (b) HUVEC cells incubated for 4 h with particles and 20 h in particle free media.
HUVEC, respectively, compared to bare MSNs. It is well known that positively charged surfaces can electrostatically interact with the slightly negatively charged cell membrane.42Therefore, the highest cellular internalization was observed for positively charged K-PA functionalized K-OMSNs. Although, both MSN and E-OMSN samples have about the same zeta potential values, around40 mV, the cellular uptake of E-OMSNs was signicantly higher than that of the MSNs. This observation indicates that the uptake rate and the amount of the nano-particles cannot be simply correlated with the net surface charge of the surface; instead, it is more related to the chemical structure of the surface functional groups.
Conclusions
In summary, we developed a straightforward method to func-tionalize MSNs with short peptide chains by using self-assembly of PAs around the hydrophobic MSNs. The resulting peptide functionalized particles can be easily dispersed in the aqueous media. We observed that peptide functionalized MSNs are not toxic to the A10 cells and HUVEC up to a high dose of 100mg mL1. In addition, it was observed that the cellular uptake of MSNs can be enhanced up to 6.3 fold by coating them with PAs, especially positively charged ones. We believe that the facile method demonstrated in this study can be applied to prepare MSNs with diverse functionalities including cancer cell target-ing, cell penetrating properties and responsive drug release, by simply changing the peptide signal to achieve selective bioactive properties.
Acknowledgements
M.S., D.M. and A.Y. are supported by TUBITAK-BIDEB fellow-ships. FACS analyses were performed at Diskapi Yildirim Beyazit Training and Research Hospital, and we thank E. Cal-isir, Prof. T. Delibasi and ADAcell for help in FACS experiments. M.O.G and A.B.T. acknowledge support from the Turkish Academy of Sciences Distinguished Young Scientist Award (TUBA-GEBIP) and TUBITAK 112T042.
Notes and references
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